Image Anal Stereol 2003;22:81-89 Original Research Paper
MORPHOLOGICAL QUANTIFICATION OF AORTIC CALCIFICATION FROM LOW MAGNIFICATION IMAGES
J esus A ngulo1, T hao N guyen -K hoa2, Z iad A. M assy2, T ilman D rueke2 and Jean Serra1
Centre de Morphologie Mathematique, Ecole des Mines de Paris, 35, rue Saint-Honore, 77305 Fontainebleau, France, 2Laboratoire INSERM U 507, Hopital Necker, 161, rue de Sevres, 75015 Paris, France e-mail: {angulo,serra}@cmm.ensmp.fr,massy@necker.fr
(AcceptedMay 14, 2003)
ABSTRACT
Atherosclerotic and medial vascular calcifications are frequent in chronic renal failure patiens and predict their increased cardiovascular mortality. Experimental models for mice have been recently developed in order to study these disorders. The aim of this paper is to present the morphological image processing algorithms developed for the semi-automated measurement of calcification from sections of aorta stained using von Kossa’s silver nitrate procedure and acquired at low magnification power (x2.5) on colour images. The approach is separated into two sequential phases. First, the segmentation is aimed to extract the calcification structures and on the other hand to demarcate the region of the atherosclerotic lesion within the tissue. The segmentation yields the image data which is the input to the second phase, the quantification. Calcified structures are measured inside and outside the lesion using a granulometric curve which allows the calculation of statistical parameters of size. The same operator computes the shape of the lesion. The relative proportion of the area of calcification is also calculated respectively for the atherosclerotic lesion area and the area outside such lesions. In conclusion, the here developed method allows quantification of vascular calcified deposits in mouse aorta. This method will be useful for the quantitative assessment of pathological vascular changes in animals and man.
Keywords: aortic calcification, automation in bioimaging, low magnification histology, mathematical morphology, nephrology, quantitative image analysis.
INTRODUCTION
Atherosclerotic and medial vascular calcifications are frequent in chronic renal failure patients and predict their increased cardiovascular mortality. Experimental models for mice have been proposed in order to study these disorders (Muntzel et al, 2002). The detection of coronary artery calcium, using mainly fluoroscopy, has been shown to be of value in predicting artery disease (Margolis et al, 1980; Reinmuller and Lipton, 1987; Tanenbaum et al, 1989). Recently, electron beam tomography has been introduced for the detection of coronary calcium (Agatston etal, 1990; Morgan-Hughes etal, 2002).
The purpose of the present work is to propose an image-based method of quantification for the uremia-enhanced vascular calcification in the aortic root, which allows the evaluation of previously established biological models.
The   images  were  derived  from  thin   sections of aortic  tissue.   The   aim  is  the  extraction  and quantification of calcification deposits. Confirmation of the presence of calcification is provided by staining using von Kossa’s silver nitrate method which is
widely used for detection of calcification (Lardenoye
etal, 2002).
In previous studies, mathematical morphology has shown its ability for solving bioimaging problems from low magnification power images in haematological cytology (Angulo and Flandrin, 2003). Such morphological approaches have also been applied successfully to other issues in nephrology (Moreso etal, 1994; Seron et al, 1996).
MATERIALS AND METHODS
STAINED AORTIC LOW MAGNIFICATION IMAGE
The model of apolipoprotein E gene knockout mice has been initially created by homologous recombination in embryonic stem cells. This genetically engineered mouse generates atherosclerotic lesions within weeks after birth that are similar to those found in humans (Zhang et al, 1992; Ishibashi etal, 1994). We created chronic renal failure in these mice by cortical electrocauterization in one kidney and ablation of the contralateral kidney
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two weeks later. After sacrifice, the aortic arch was cut into serial sections in a cryostat. Sections were stained with von Kossa’s silver nitrate method. The animals were treated according to the recommendations of animal care committees, under anesthesia (Protocol: short term anesthesia of a duration of 30-40 min; one IP injection composed of 75 ju.l Rompun 2% in 25 ml vial, 300 ju.l Ketamine CLORKETAM 1000 in 10 ml vial, 1.53 ml 0.09% NaCl; the amount of the anesthetic fluid administered 100 — 120 jlil / 20 g body weight).
Fig. 1. Images under two magnification powers (x.2.5 and x10) of a section of the aortic sinus showing the elements of interest: tissue, atherosclerotic lesion and calcification structures.
In Fig. 1 are depicted two colour images under two magnification powers (x2.5 and x10) of a section of the aortic sinus showing the elements of interest: tissue, atherosclerotic lesion and calcification structures. In this study, the series of colour images were acquired under very low magnification power at x 2.5. The main advantage is that in one image field all the tissue to be examined is included; however there is a considerable drawback since the quality of the structures is mediocre. Therefore the automation of the segmentation and quantification procedures involves the development of specific algorithms using advanced techniques of image analysis. This is the rationale behind the use of mathematical morphology operators. As one can see from Fig. 1, other alternatives are possible. For instance, working at the magnification power of x10 and using a motorized microscope is an alternative to acquire several subimages to cover the whole area of interest. This approach has however serious drawbacks including definition of lesion at x 10, extraction of the whole calcification region and merger of quantified data (overlapping of images).
The size of the image is 672 x 538 pixels on a rectangular raster with 256 grey tone levels for each red, green and blue colour channels. We consider for quantification that at x2.5 magnification and for the resolution used, the size of a pixel is 5.5 um2/pixel.
(a)
(b)
(c)
(d)
(e)
Fig. 2. Example of microscopic field image from a section of aorta stained using the von Kossa method at low magnification power (y.2.5): (a) RGB colour image fRGB, (b) red component fR, (c) green component fG, (d) blue component fB, (e) saturation component fS.
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Fig. 2 is an example of colour image to be processed, frgb. The red fr, green fG and blue fB colour component images are shown and beside this, the saturation component fS of a 3D-polar coordinate colour representation (Hanbury and Serra, 2002). For a colour pixel c = (r,g,b); i.e. frgB(c) = Cfr(c),fg(c),fb(c)) the saturation coordinate fS(c) is obtained by the simple expression
fS(c) = max(r,g,b) -min(r,g,b).           (1)
Using the spectral properties of the staining procedure on the tissues (calcifications appear black and tissues red-pink), we use the colour component images in the following way:
-    The calcification is relatively more contrasted against the rest of tissues in fR, therefore this component is used for extracting the calcifications.
-    In order to detect the tissue in the field image the simplest means is to take fS where the biological structures are clearly defined, despite the shading effect (non-uniform illumination).
-    For segmenting the region of the atherosclerotic lesion a combined method of thresholding and manual interaction is achieved from fG.
Through this choice of the colour components, the color shading effect is negligible. Anyway, a method for shading correction of color images (Tomaz¡evic¡ et al, 2002; Derganc et al, 2003) can be used in applications with important multispectral inhomogeneities.
MORPHOLOGICAL IMAGE PROCESSING AND QUANTIFICATION
First introduced as a shape-based tool for binary images, mathematical morphology has become a very powerful nonlinear image analysis technique with operators capable of handling sophisticated image processing tasks in binary, grey-scale, colour and multiresolution imaging modalities. A tutorial in the technique can be found in (Serra, 1982, 1988; Coster and Chermant, 1989). In this section we briefly review the basic morphological operators used in this work.
In the framework of digital grids, a grey-tone image can be represented by a function f : Df —> T, where Df is a subset of Z2 and T = {tmin,..., tmax\ is an ordered set of grey-levels. Let B be a subset of Z2 and s G N a scaling factor. sB is called structuring element (shape probe) B of size s. The basic morphological operators are:
-    dilation: dB(f(x)) = supyeB{f(x - y)}
-    erosion: eB(f{x)) = inf_yeB{f(x-y)}
The two elementary operations of erosion and dilation can be composed together to yield a new set of operators having desirable feature extractor properties which are given by:
-    opening: gB(f) = dB[eB(f)}
-    closing: jB(f) = eBdB{f)]
The morphological openings (closings) filter out light (dark) structures from the images according to the predefined size and shape criterion of the structuring element.
The top-hat transformation is a powerful operator which permits the detection of contrasted objects on non-uniform backgrounds (Meyer, 1977). There are two versions:
-    white top-hat: The residue of the initial image f and an opening g{f); i.e. r(f)= f— gf), extracts bright structures,
-    black top-hat: The residue of a closing j(f) and the initial image f;r(f) = j f) — f, extracts dark structures.
Usually, the top-hat is accompanied by a thesholding operation, in order to binarise the extracted structures.
A granulometry is the study of the size distributions of the objects of an image. Formally, a granulometry can be defined as a family of openings G = g n)n>0 such that n > 0,m > 0,g n gm = gmg n = gmax(n,m). Moreover, granulometries by closings (or anti-granulometry) can also be defined as families of increasing closings F = j n)n>0. Performing the granulometric analysis of an image f with G is equivalent to mapping each opening of size n with a measure J%gn{f)) of the opened image. M{f) is the area of f in the binary case (number of pixels) and the volume in the grey scale case (sum of pixel values). The size distribution or pattern spectrum of f with respect to G, denoted PSG(f) is defined as the following (normalised) mapping
PSG(fn)=PS(fn) =
J?gn(f))-JZgn+1{f))
---------------—-------------,    n > 0 .    (2)
m{f)
The pattern spectrum PSG(f) maps each size n to some measure of the bright image structures with this size (loss of bright image structures between two successive openings). The pattern spectrum PSG(f,n) is a probability density function (a histogram): a large impulse in the pattern spectrum at a given scale indicates the presence of many image structures at that
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scale. It is also possible to use standard probabilistic definitions to compute the moments of PS. The first moment m is given by m = ml = Y„nPS(fin), the k-th pattern spectrum moment, k > 2, is computed as mk = X„(n — mkPS(f,n). In particular, the first four moments: mean m, variance m2, skewness m3 and kurtosis mA are often used.
Using a pair of an opening g and a closing j as primitives, an operator of contrast enhancement can be obtained (Serra, 1989). This toggle mapping or two states contrast k is generated by the following criterion applied to each point x,
r   j) if
<p(fx)-f(x)>fx-gfx)
kf(x))-\ j j      if
K{J{)} ~ |     <p(fx) -fx < fx - gfx)
fx   if
I    jfx)-fx = f(x)-gfx)
(3) The closing and the opening may be replaced by a dilation and an erosion off.
A morphological tool that complements the opening and closing operators for feature extraction (extract the marked particles) is the morphological reconstruction, implemented using the geodesic dilation operator based on restricting the iterative dilation of a function marker f by B to a function maskg, d»{f) = dldng-\f), where d*(f) = dB(f)Ag. The reconstruction by dilation is defined by
rec(g,f) = d-(f)                   (4)
such that dg(f) = dg+l (f) (idempotence).
The thresholding transformation of the image f between the grey levels u\ and u2 (typically u\ = tmin or u2 = tmax), TMli„2] (f), yields the binary image I such that for each pixel x the binary value is given by
Ix = T     2f-x) = {1   u h~erwise-u2-     (5)
The choice of \u\,u2\ determines the set of grey levels associated with the object of interest. The histogram summarises the grey-level contents of an image and typically, the optimal threshold values can be obtained from an analysis of the histogram, see in (Angulo and Flandrin, 2003) the method of automated thresholding which combines the classical selection of the threshold value by minimising the sum of within class variances with a morphological technique for selecting the central mode values which speeds up
the convergence to the u’s. An alternative to make the choice of [ui,u2] easier is a method which relies on a double thresholding combined with a geodesic reconstruction; the technique is known as thresholding by hysteresis (Soille, 1999). For instance, in order to extract light structures we have to take u2 = tmax and let uT,    be a low threshold value and uT.., be a
± low                                                                                                           -1 high
high threshold value. Using this double threshold, two binary images are obtained: Iwlde = T[uT^tmax](f) and I narrow = T[uThigh,tmax] (f). The final binary image is given by the reconstruction ofIwide using Inarrow as a marker; i.e.
Tut    Out      t     ]\f ) = g       InarrowiIwide)-        (6)
ALGORITHMS
The approach is separated into two sequential phases. First, the segmentation is aimed on the one hand to extract the calcium structures and on the other hand to demarcate the region of the lesion on the tissue. The segmentation yields the image data which are the input to the second phase, the quantification. The calcification structures are measured inside and outside the lesion using a granulometric curve which allows to calculate some statistical parameters of size. The same operator is used to compute the shape of the lesion. The relative proportion of area of calcification is also calculated.
Detection of tissue in the field image
We start by binarising the saturation component fs for detecting the tissue presented in the field image by using a simple thresholding operation at uTl (a low value; e.g. uTl = 10, has shown to be suitable for this kind of images), see Fig. 3(a),
I = Twri,w]Cfs)-                       (7)
In order to remove the noise and mistakes due to the small pieces of tissue (histology artefact), the image is then filtered by applying a reconstruction using an opening as the marker,
Itissm = geCgsAIs),Is),                         (8)
with si such that s^B is larger than the size of the noise and the small artefact pieces of tissue (good results were obtained with si = 5); the structuring element B is a circle. See the result in Fig. 3(b).
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Fig. 3.   Detection of tissue  in the field image: (a)   Binary   image   after   thresholding   saturation component, Is; (b) cleaned binary mask of tissue, ItiSSUe.
Interactive segmentation of the region of atherosclerotic lesion
In the tissue, the atherosclerotic lesion zone squares with a texture of tissue less thick than the rest, recognising visually, but its precise morphological definition is almost impossible. Therefore due to the fact that the automated segmentation of the lesion on the tissue can not be achieved without some mistakes and in order to avoid the propagation of these errors to the quantification step, we preferred to developed an interactive approach.
In a first step the human expert must perform a manual dot-marking of the region of interest (ROI) using the computer mouse on the colour image. This zone associated to the lesion has to be closed, see Fig. 4(d), but it is possible for the human user to demarcate several closed partial ROI’s: the definitive ROI is the union of the partial ones and is represented by the binary image Imrk.
The automated step works on the green component fa. This image is simplified by means of an opening by reconstruction (which simplifies the light structures),
fG = gecgsAfo)fo),              (9)
Fig. 4. Interactiv segmentatio of the region of lesion: (a) Simplified green component using an opening by reconstruction, fG; (b) followed by closing by reconstruction, fl; (c) negative of the binary mask without empty image zones, Ig; (d) manual dot-marking region of interest, Imrk; (e) binary mask of lesion inside the region of interest, IieSion.
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Angulo J et al: Morphological quantification of aortic calcium
followed by a closing by reconstruction (which simplifies the dark structures),
ß = jrecjsAfG)fG),            (10)
where the sizes of the opening and the closing have been empirically fixed to s 2 = 15 and s3 = 10 respectively (B is a circle). The corresponding example of tissue image after simplification is shown in Fig. 4a - b. The image f" G must be binarised for extracting the empty zones (zones of the field without tissue); the optimal threshold value uTl for each image is obtained by the algorithm presented in Angulo (02). The result of this thresholding process at uTl,
Ig = T[tmimuTl}{fa)^                          (11)
is a first binary mask which is then restricted to the manual defined I,rk to obtain the binary mask of the region of lesion,
Ilesion       Imrk 'Ig*                         (12)
See the result in Fig. 4e. Extraction of calcification
In the red component of the aortic section colour image fR the calcium appears as dark structures upon a bright background. In order to enhance the contrast of the calcifications against the background a toggle mapping is taken,
fr = kfR),                      (13)
where the two primitives of k are an erosion and a dilation of size 3, see Fig. 5(a). From this image, a black top-hat of size s4 extracts the calcification (s4 corresponds to the size of the biggest calcium structures which can be found); experimentally we have fixed s 4 = 25 with a circular structuring element,
fc = r{fR).                     (14)
On the image fc, Fig. 5(b), a thresholding by hysteresis is performed to provided the binary mask of calcifications (see Fig. 5(c)),
Icalcif = T[Irlm<>uT high,tm\ f);                (15)
the choice of the threshold values is not so critical (the top-hat facilitates just the thresholding); e.g. uTlow = 50
Fig. 5. Extraction of calcification:   (a)    Contrast enhancement of calcification on red component, fR; (b) extracted calcification by means of a dual top-hat, fc; (c) binary mask of calcification after thresholding by hysteresis, ICaiaf.
Quantification of atherosclerotic lesion
Let srf{I) be the surface area; i.e., number of pixel to ones, of the binary image I. We start by calculating the size of the tissue and the lesion regions,
Atissue        ™ \Iissue) i      Alesion        ™ \Iesion) •       (16)
We propose to characterise the shape of the lesion region by means of a pattern spectrum curve, PS(Iiesion,n) = PSiesion. The structuring elements are circles of increasing size n + 2 (in fact, the practical shape is an octagon: isotropic approach to a circle in the square grid), from n = 4 to n = 70. The granulometric curves describe in a compact way the different thickness of the wall of the aortic valves: each
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peak corresponds to the thickness of a sector of the wall. These histograms of shape can be parametrised by using their statistical moments. The surface area in pixels of an octagon of size n is given by the formula
(Serra,  1982), Nc
octagon
7n2 + 4n + 1. In order to obtain the sizes in (im2 we have to multiply the area in pixels by 5.5 (im2/pixel for all the measurements.
Quantification of calcification
In order to quantify the calcium, we compute first the absolute and the relative surface of calcifications inside and outside the lesion,
Acalci/J A calci /jo
calci/' f\Ilesion) •>
calci/ '\ \Itissue      Iesion)) i             (17)
Acalci/J
Acalci/jo
Acalci/J Ilesion)
A calci/jo
&y \Itissue      Ilesion)
(18)
(19)
Besides these parameters, the calcification structures inside the lesion are quantified by using a size distribution,   PS{Icaicif A I/
esionin)    —   PScalcifJ     (the
structuring elements of openings are again circles of increasing size n + 2, from n = 3 to n = 23) and its moments.
RESULTS AND DISCUSSION
In Fig. 6 two examples of segmented aortic section images are depicted: one corresponding to a mouse with low calcification content (control mouse) and another to high calcification (uremic mouse). In Fig. 7 their associated pattern spectra are shown.
In Table 1 the obtained parameters of the lesion are included. For mouse #35 the area of lesion is larger than for mouse (J40, but above all, the thickness of the walls is considerably greater. Besides being used for determining the relative amount of calcium located inside, the size of the atherosclerotic lesion may be significant a priori. However, due to the fact that during the tissue preparation procedure the shape of the atherosclerotic lesion may be modified involuntarily, the practical usefulness of the parameters from PSiesion remains doubtful. Other techniques of tissue preparation could be envisaged in order to achieve more reproducible sections in such a way that the parameters associated to PSiesion will be very important.
¦¦„-. :	L      M
jA	1 p-1
».'.j    j	l__-«* v™
Fig. 6. Two examples of aortic section images: (a) with low calcification content (mouse 135); (b) riches in calcification (mouse (140). On the left, the initial images and on the right, summary of segmented structures: the background in black, the tissue in gray, the lesion in white and the calcification in red.
Pattern spectrum of
Size distribution of calcification
Mouse 35 Mouse 40
(b)
Fig. 7. Quantification of size and shape using morphological granulometries (curves associated to the examples of Fig. 6): (a) Pattern spectrum of the region of lesion; (b) Size distribution of calcification structures inside the lesion.
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Angulo J et al: Morphological quantification of aortic calcium
Table 1.  Size (Atissue and A[esion) and shape (first four moments ofPSiesior) parameters of lesion zone for the examples of Fig. 6.
Atissue       Alesion          m             m                 m                     m
Mouse 35     120859    59079    38.55    293.03    -1176.89    208019.51 Mouse Ü40     160483    78696    51.17    647.21     -206.51     720299.94
Regarding the parameters of calcification, summarised in Table 2, the use of this set of parameters yields an easy way to identify the different levels of calcification. Obviously, the most interesting parameters are the relative surfaces of calcification and the two first moments of size distribution: the mean size of calcium structures and the variance of size (which gives an idea of size dispersion and is helpful for distinguishing large compact calcifications from large disintegrated calcifications).
Table 2. Surface area parameters and first four moments of PScaicifj of lesion zone for the examples of Fig. 6.
Acalcifj      Acalcif-D      Acalcifj      Acalcif-o
Mouse t)35       821          170        0.0139     0.0014
Mouse tt40     43094      37137      0.5476     0.2314
(a)
	fl	ßZ	H6	M4
Mouse (J35 Mouse (J40	2.19 5.59	0.48 12.92	0.39 74.21	0.32 729.99
(b)
The robustness of an algorithm can be defined with respect to changes in the parameters or to image quality. The present algorithms have seven configurable parameters: ur, (threshold value for the tissue determination on fs), s1 (size of the tissue filtering on fs), s2 and s3 (size of the tissue simplification on ff), s4 (size of the calcium enhancement on fR), uT,   and uT,.. (threshold values
J 1 v' "       ± low                      -1 high    v
for the calcium extraction on fR). The threshold value uTn is obtained automatically for each image. The values proposed for the other parameters have been set after empirical tests on a random selection of 10 images which covers the different levels of pathology and image quality. Someone who would like to use these methods with a different magnification or a different camera has to start by using a training set of images (representative of its problem) in order to adjust the values of parameters. In any case, some tests of images at x 10 have shown that the approach is quite robust.
Once these parameters were fixed, a deep study has been performed on the basis of the present methods. A database of 173 images corresponding to 45 mice
has been used. The results of the interactive human segmentation of the atherosclerotic lesion and the extraction of calcification have been subjected to evaluation by another human grader in order to find out serious mistakes: only 3 cases among the set of 173 have been rejected and manually corrected (1.7%). In view of these results we can state that the behaviour of the image analysis algorithms is quite satisfactory.
The approach has however several limitations. On the one hand, in order to minimise the errors of segmentation, an interactive algorithm was developed which involves a necessary human action for each image. Obviously, the task is simple (some “clicks” of mouse) but time consuming. The balance between automation and precision leads sometimes to this kind of approach. On the other hand, working on low magnification microscopic images entails that the image structures can be very small, limiting the resolution of the methods. For instance in Fig. 7 (right), notice that the size distribution of calcium for the control mouse (small calcification structures) is limited to the openings of size n = 3 and 5. The classical problem of reproducibility of histologic preparations (sectioning, staining, etc.) must be also taken into account in order to prevent artefacts, like dust or others.
CONCLUSION
The robustness and accuracy of segmentation results allowed us to consider the subsequent quantification as a correct procedure. The morphological parameters obtained have been analysed and correlated to several biomedical parameters (Massy et al, 2003). We also envisage to apply this quantification approach to the study of calcification using non-invasive techniques, for instance by obtaining the morphological information by means of electron beam tomography or other imaging technique. In the previous studies based on computed tomography (Agatston et al, 1990, Morgan-Hughes et al, 2002), the quantification of calcification has not been attempted using morphological techniques.
To summarise the algorithmic discussion above, we   conclude   that   quantitative   measurements   of
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histologic sections by morphological image analysis software are very useful. They provide a powerful tool for improving and automating experimental studies on structural-functional correlations in tissues with histopathologic changes.
ACKNOWLEDGMENTS
The authors thanks the comments and suggestions of the anonymous reviewers.
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